Intracellular Ca 2+ signaling and the regulation of vesicular exocytosis are two fundamental physiological properties of all eukaryotic cells. They have been analyzed in detail in only a few exemplar cell types. We need precise descriptions in each cell type to understand the implications for disease and therapy. This project will study pancreatic beta-cells, pancreatic ductal epithelium, chromaffin cells, pituitary gonadotropes, and sympathetic neurons. It will use patch clamp biophysical methods and optical Ca 2+ reporters to quantitate sources and sinks of Ca 2+ in differentiated mammalian endocrine, nerve, and epithelial cells. One focus will be on Ca 2+ buffering and Ca 2+ clearance. In these cells, cytoplasmic buffering and four membrane clearance processes shape the Ca 2+ transient and thus regulate secretion of the endocrine hormones insulin, adrenaline, and gonadotropins and the secretion of mucus of the gastrointestinal tract. Defects in regulation of secretion underlie some forms of diabetes, infertility, cystic fibrosis, and digestive disorders. We need basic understanding to inspire new therapeutic approaches. This project will determine a kinetic model for the secretory vesicle pools of pancreatic ductal epithelium, including their regulation by Ca 2+, protein kinases, and other physiological variables. The Ca 2+ buffering and Ca 2+ clearance mechanisms of sympathetic neurons, pancreatic beta-cells, and pancreatic ductal epithelial cells will be dissected and described by a quantitative model. Our analysis of the Ca 2+ dynamics within the endoplasmic reticulum of gonadotropes and within the mitochondria of chromaffin cells will be deepened. What are the Ca 2+ buffering and flux properties of these organelles? Our work in these cells concerns processes whose failure leads to disease and whose modulation offers new therapies.